Antimicrobial resistance, Extended-Spectrum β-Lactamase production and virulence genes in Salmonella enterica and Escherichia coli isolates from estuarine environment

The impact of antimicrobial resistance (AMR) on global public health has been widely documented. AMR in the environment poses a serious threat to both human and animal health but is frequently overlooked. This study aimed to characterize the association between phenotype and genotype of AMR, virulence genes and Extended-Spectrum β-Lactamase (ESBL) production from estuarine environment. The Salmonella (n = 126) and E. coli (n = 409) were isolated from oysters and estuarine water in Thailand. The isolates of Salmonella (96.9%) and E. coli (91.4%) showed resistance to at least one antimicrobial agent. Multidrug resistance (MDR) was 40.1% of Salmonella and 23.0% of E. coli. Resistance to sulfamethoxazole was most common in Salmonella (95.2%) and E. coli (77.8%). The common resistance genes found in Salmonella were sul3 (14.3%), followed by blaTEM (11.9%), and cmlA (11.9%), while most E. coli were blaTEM (31.5%) and tetA (25.4%). The ESBL production was detected in Salmonella (1.6%, n = 2) of which one isolate was positive to blaTEM-1. Eight E. coli isolates (2.0%) were ESBL producers, of which three isolates carried blaCTX-M-55 and one isolate was blaTEM-1. Predominant virulence genes identified in Salmonella were invA (77.0%), stn (77.0%), and fimA (69.0%), while those in E. coli isolates were stx1 (17.8%), lt (11.7%), and stx2 (1.2%). Logistic regression models showed the statistical association between resistance phenotype, virulence genes and ESBL production (p < 0.05). The findings highlighted that estuarine environment were potential hotspots of resistance. One Health should be implemented to prevent AMR bacteria spreading.


Introduction
Antimicrobial resistance (AMR) has been recognized as one of the greatest challenges endangering the health of people, animals, and the environment. One Health approach has been applied for managing and controlling AMR at national and international levels. The Unites States Center for Disease Control and Prevention (U.S. CDC) estimated that greater than 2.8

Detection of AMR gene
All isolates were tested for the presence of AMR genes including genes represented to ampicillin (bla TEM ), chloramphenicol (catA, catB and cmlA), quinolone (qnrA, qnrB, and qnrS), aminoglycosides (acc(3)IV and aadA1), streptomycin (strA and strB), tetracycline (tetA and tetB), sulfamethoxazole (sul1, sul2, and sul3), and trimethoprim (dfrA1 and dfrA12) ( Table 1). Conventional PCR was performed to detect most AMR genes, except genes corresponding to quinolone and sulfamethoxazole, which were used multiplex PCR. DNA templates of all E. coli and Salmonella were prepared using whole cell boiling technique [17]. Toptaq PCR Master Mix Kit (Merck, Munich, Germany) were followed as manufacturer's instruction. The PCR products were separated by gel electrophoresis using 1.5% agarose gel in 1X Tris-acetate/EDTA. Gels were stained with Redsafe™ Nucleic Acid Staining Solution (iNtRon Biotechnology, Seongnam, South Korea) and visualized PCR products under UV light using Omega Fluor™ gel documentation system.

Phenotypic and genotypic detection of ESBL production
Disk diffusion method was used to examine ESBL production followed by CLSI standard [16]. The detection of ESBL production consists of screening and confirmation tests. Ceftazidime (30 µg), cefotaxime (30 µg), and cefpodoxime (10 µg) were used for initial screening. All isolates that showed resistance to at least one of cephalosporins were further confirmed using a combination disk diffusion method using cephalosporins combination with clavulanic acid. The positive ESBL production was interpreted by determining the difference of inhibition zone between solely cephalosporin and cephalosporin combine with clavulanic acid. The positive ESBL-production isolates were identified β-lactamases genes (bla TEM , bla SHV , bla CMY-2 , and bla CTX-M-55 ). The bla TEM gene was examined using conventional PCR, while bla SHV , bla CMY-2 , and bla CTX-M-55 were using multiplex PCR with the specific primers as described in Table 2.

Nucleotide sequence
PCR amplicons of positive ESBL production isolates were purified using GeneJET PCR purification kit (Thermo Fisher Scientific, Vilnius, Lithuania) and submitted for DNA sequencing (Bionics Co., ltd., Gyeonggi-Do, Republic of Korea). The result of the DNA sequence was blasted and aligned with references embedded in GenBank database available from the National Centre for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/ BLAST) (accession number OQ282894-OQ282896).

Detection of virulence genes
Virulence genes of Salmonella, including invasin (invA), fimbrial protein (fimA), and enterotoxin (stn) genes were observed (Table 3). Heat-labile toxin (lt), heat-stable toxin (st), STEC (stx1 and stx2) and EPEC for attaching and effacing protein (eae) were examined in all E. coli isolates. Most of virulence genes were detected using conventional PCR. The detection of stx1 and stx2 genes was performed by multiplex PCR.

Statistical analyses
Descriptive statistics were performed to identify prevalence of resistance phenotype and genotype, resistance pattern, MDR, virulence genes, and ESBL production of E. coli and Salmonella isolates. Logistic regression analysis was used to examine the association among AMR, virulence genes and ESBL production. The dependent variable was the highest resistance rate, and independent variables included resistance genes, resistance phenotype, virulence genes, ESBL production and MDR. A p-value and confidence intervals of regression analyses were adjusted for potential correlated data within type of sample (oysters and estuarine waters) using robust variant estimator. Univariate analysis was performed to screen for potential significance of predictors. Forward selection and backward elimination were used to select potential candidates for multivariable analysis. Final regression models of E. coli and Salmonella were received based on p < 0.05 and likelihood ratio test. All statistical analyses were performed using Stata 14.0 (StataCorp, TX, USA). Two-sided hypothesis tests were used with 5% of significant level.

Co-existence among AMR, ESBL production, and virulence genes
One Salmonella isolated from oyster sample harbored bla TEM-1 with MDR to ampicillin, chloramphenicol, sulfamethoxazole, trimethoprim, and tetracycline. The latter was also positive to invA, sul3, cmlA, and dfrA12 genes. An ESBL-producing E. coli isolated from cultivation water harbored bla TEM-1 was resistant to ampicillin, while three ESBL-producing E. coli with bla CTX-M-55 were MDR.

Discussion
One of the main findings of this study is more than 90% of Salmonella and E. coli from fresh oyster (96.7%; n = 119/123 of Salmonella and 95.2%; n = 238/250 of E. coli isolates) and estuarine water (100.0%; n = 3/3 of Salmonella and 92.5%; n = 147/159 of E. coli isolates) samples were resistant to at least one antimicrobial. MDR Salmonella (23.0%) and E. coli (40.0%) were also isolated, even though the oysters were received from wild caught with no evidence of antimicrobial use. This cultivation site could be contaminated from nearby communities and agriculture according to previous studies [29,30], so that trackback investigation to identify the source of AMR in estuarine environment is recommended. Estuarine water was considered a potential hotspot to surveillance of AMR distribution in the environment [30]. Humans can be infected with AMR bacteria by eating aquatic animals or direct contact with contaminated environment. Resistant bacteria found in this study are considered as important estuarine environmental pollutants that can adversely affect food security and public health. In this study, the highest resistance rates acquired by both Salmonella (95.2%) and E. coli (77.8%) was to sulfamethoxazole. However, a previous study reported the lower prevalence of Salmonella resistant to sulfonamides (56.5%) in retail aquaculture products such as shellfish, calm, fish, shrimp, and others in Shanghai [31]. The high prevalence of sulfamethoxazole observed in this study may be widely used in human and animal medicine because this antimicrobial can be used to treat and prevent many bacterial infections at affordable cost [32].Therefore, it is possible that sulfamethoxazole could disseminate and accumulate to the environment. Sulfamethoxazole is effective against both Gram-negative and Gram-positive bacteria, including E. coli and Listeria monocytogenes. This antimicrobial agent is commonly used to treat urinary tract infection, bronchitis, and prostatitis. In veterinary medicine, sulfonamides have been used in swine and cattle production for treatment of urinary and respiratory tract infection. High concentration of sulfonamides in the environment has been indicated in livestock manure due to the common use of this antimicrobial [33,34]. Sulfamethoxazoleresistant bacteria was also found in surface water and soil causing environmental pollutants as a result of the widely used in treatment of animals and humans [35]. The impact of sulfonamides contamination in the environment could result in hazardous to human health (e.g., difficult to treat of resistant bacteria, prolong hospital stay etc.), and alter microbial community [33]. However, the consequences of sulfonamide contamination in the ecosystem were still unclear [36]. Therefore, the removal of these resistant bacteria from healthcare facilities, livestock farms, and communities are needed to reduce the contamination to the coastal environment. Previous studies developed the removal of sulfonamides by using anaerobic membrane bioreactor in swine wastewater, and the use of Pleurotus eryngii for degradation of sulfonamides [37,38].
Besides sulfamethoxazole resistance, the high resistance was observed for trimethoprim (37.3%) and ampicillin (36.5%) in Salmonella, and for ampicillin (55.3%), and tetracycline (40.1%) in E. coli. These findings agreed with previous studies conducted in aquatic animals and estuarine environment [39,40]. High resistance rates to ampicillin (100%) and erythromycin (83.33%) in Salmonella isolates were previously reported in water and sediment [41]. The high resistance to sulfamethoxazole, ampicillin, tetracycline, and trimethoprim observed in this study was commonly reported in humans and animals [42][43][44][45]. In Thailand, the molecular epidemiology and association of AMR among of E. coli and S. enterica have been extensively investigated from pigs, pork, and humans indicating the potential risk of AMR spreading [43,46]. Even though the precise genetic relationship information is still lacking, the observations of resistance to these antimicrobials in humans, food-producing animals, and environment in the same country confirm that AMR is a complex One Health issue.
S. enterica serovar Paratyphi B causes a serious disease, Paratyphoid, in humans. The serovars Paratyphi B poses a significant health risk due to being associated with sporadic outbreaks of human infection and multistage outbreaks of seafood products [46][47][48]. The symptoms of paratyphoid infection in humans are fever, loss of appetite, weakness, headaches, diarrhea, and may be a life-threatening multi-systemic illness. The pathogens were recently isolated from poultry and poultry meat from Europe and Latin America [49]. A study reported that serovar Paratyphi B was isolated from oysters (22.7%) in Thailand [15]. In this study, the serovars Paratyphi B was isolated from oysters (13.5%, n = 17/126), and all these isolates were resistant to at least one antimicrobial and 29.4% (n = 5/17) were MDR. More than 75% (n = 13/17) of these isolates contained virulence genes (i.e., fimA and stn), and 64.7% (n = 11/17) of all Paratyphi B isolates harbored invA. The presence of MDR Paratyphi B isolates in oysters may pose a serious threat to public health in the near future due to the difficulty in controlling strategic action.
Most resistance genes detected in this study corresponded well to observed resistance phenotype, suggesting that resistance genes were usually expressed when present. In Salmonella isolates, the most detected resistance genes were sul3 (14.3%), bla TEM (11.9%), cmlA (11.9%), and tetA (11.1%), while those in E. coli isolates were bla TEM (31.5%), followed by tetA (25.4%) and strA (14.9%). High prevalence (91.3%) of bla TEM gene was previously reported in oysters [50], which agreed with this study. This study observed the presence of β-lactamase encoding bla TEM-1 indicating a narrow spectrum activity against β-lactamase of E. coli and Salmonella. This indicated that the estuarine environment serves as a potential hotspot of AMR bacteria carrying resistance determinants that may be transferred to bacterial pathogens in humans and animals.
In this study, the occurrence of ESBL-producing E. coli (2.0%) and Salmonella (1.6%) was lower than in a previous study, which greatly varied in humans (11-72%), animals (0-72%), and wastewater (7-79%) in West and Central Africa [51]. Greater than 40% of wastewater from Tunisia were positive to ESBL-producing Enterobacteriaceae [52]. In this study, bla TEM-1 (n = 2) and bla CTX-M-55 (n = 3) were reported with MDR, which agreed with previous studies in aquatic environment and migratory birds [53,54]. Furthermore, the bla TEM and bla CTX-M isolates were the common widespread genes from wild fish and aquatic environment [54,55]. More specifically, the bla TEM-1 , bla CTX-M-14 and bla CTX-M-15 genes were reported from marine bivalve mollusks [8]. Even though the low rates of ESBL producing bacteria were observed in this study, the positive ESBL isolates were commonly identified MDR bacteria. Hence, the occurrence of ESBL producing bacteria that harbored MDR signifies the public health threat.
The association between resistance to sulfamethoxazole and other predictors, including AMR, MDR, virulence genes, and ESBL production were examined under the logistic regression models (Tables 7, 8). The complexity of association among resistance and virulence of E. coli and Salmonella was observed. Sulfamethoxazole resistance in E. coli was positively associated with trimethoprim resistance, ESBL production, MDR, and the presence of addA1, strA, and sul3, but these isolates were negatively associated with lt, stx, and dfrA12. The major concern of these findings was almost half of E. coli carrying virulence genes were MDR bacteria. A co-selection of resistance and virulence can occur through mobile genetic elements such as integrons, transposons, and integrative conjugative elements [56]. The infection of resistant and virulent pathogens is detrimental to human health since they cause difficulty to treat and increase treatment failure. On the other hand, sulfamethoxazole resistance in Salmonella was positively correlated with resistance to ampicillin and trimethoprim, and invA, but they were negatively associated with ESBL production and stn. This finding indicated the complexity of AMR, virulence factors and resistance determinants in the environment. A quarter of Salmonella carrying virulence genes were MDR. Thus, sulfamethoxazole resistance isolates can co-selection to many classes of antimicrobials, virulence genes, and ESBL production. A previous study indicated that resistance and virulence plasmids were linked simultaneously [57]. As a result, the infection of resistant and virulent bacteria may cause more complicated treatment and increase morbidity and mortality rates due to failure of bacterial treatment. Shiga toxin is bacterial exotoxin related to highly cytotoxic class II ribosome [58]. In this study, stx1, Shiga toxin-producing E. coli (STEC) was most frequently found in oysters and estuarine waters, while eae gene representing enteropathogenic E. coli (EPEC) was reported in estuarine water at a low rate (0.2%). A previous study indicated that none of virulence genes related to STEC and EPEC were identified in oysters and mussels from Atlantic Canada [29], in contrast to the results in this study. Wildlife and aquaculture, including fish and shellfish have been identified as one of important sources of STEC spillover from livestock animals [59]. The high rate of stx1 in this study raise public health concerns of seafood safety, since major clinical signs of STEC infection in humans are bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome, and may be life-threatening.
The fimA, stn, and invA genes are common virulence genes that play an important role in the pathogenicity of Salmonella infection. The fimA gene is a common structural subunit of type 1 fimbrial protein, while stn is heat-labile Salmonella enterotoxin affecting epithelial cells [60,61]. The invA gene is an important structural component of Salmonella pathogenicity island, which is related to invasion of gut epithelial tissues in human and animals [28]. In this study, 77.0% of Salmonella isolates were positive to invA gene, even though this gene has been used for confirmation of Salmonella in food animals. This agreed with previous studies where the absence of invA gene was found in poultry production [62,63]. In seafood and environmental samples, some Salmonella isolates confirmed with biochemical test did not contain invA gene [64][65][66]. The absence of invA gene may be because Salmonella was not invasive or had other invasive mechanisms [67]. However, the absence of invA genes is a rare occasion. The combination of PCR and next generation sequencing (NGS) is proposed to increase sensitivity of Salmonella detection of resistance in environmental samples [68].
In conclusion, MDR and ESBL-producing E. coli are widespread in the estuarine environment, highlighting the need for continuing AMR monitoring programs in shellfish harvested area. Knowing the magnitude of AMR circulated in the environment can facilitate developing strategic action plans to mitigate the possible transmission of resistance bacteria among humans, animals, and environment. In addition to phenotypic detection of AMR, identification of AMR driving sources and monitoring of genetic information of resistance organisms are required to better understanding reduce the occurrence and transference of AMR in aquatic animals and estuarine waters. Oysters and estuarine water serve as overlooked natural reservoirs of AMR contamination. Awareness of seafood safety and increase personal hygiene are suggested to reduce AMR infection from seafood consumption.

Acknowledgments
The author would like to thank Saran Anuntawirun for laboratory assistance.